PROJECT SUMMARY The most important prognostic indicator following cancer surgery is complete resection. Intraoperative detection of tumor margins is challenging, and residual disease continues to be the most common cause of local recurrence. Complete resection results in prolonged patient survival and improved post-surgical quality of life. Visual enhancement of tumors using near-infrared (NIR) imaging with targeted fluorophores can identify lesions that are not detectable by visual observation or palpation. NIR imaging offers high resolution and sensitivity and can be performed in real time in an intraoperative setting. This application aims to test the efficacy of targeted NIR fluorophores for tumor detection in murine models of lung cancer and to translate these findings to guide intraoperative surgical tumor removal in canine patients with spontaneous lung cancer. Lung cancer is the leading cancer-related cause of death and the third most diagnosed cancer in the United States. In non-small cell lung carcinomas (NSCLC), which represent 90% of clinical lung cancers, tumor resection continues to be the most effective approach to cure patients with local disease. Surgeons typically use visual inspection and finger palpation to define solid tumor margins. However, this approach is often insufficient for the detection of residual disease, thus leading to locoregional recurrence in up to 40% of patients and significantly reduced 5-year survival in this cohort. In this proposal, we will use NIR probes synthesized in our laboratory and designed to detect choline kinase (ChoK), a lipid kinase upregulated early in tumorigenesis and overexpressed in 56% of lung cancers and up to 83% of NSCLC. These probes will be tested for their ability to detect tumor margins in two models of NSCLC in mice. We will evaluate time to tumor recurrence and survival in mice after fluorophore-targeted and non- targeted surgical excision. From these data, one fluorophore will be selected for synthesis under GLP conditions and its systemic toxicity evaluated in a cohort of canines. We will then recruit a patient cohort of 30 companion animals presenting to the University of Pennsylvania Veterinary School with spontaneous NSCLC. Our Preliminary Studies indicate that canine NSCLC significantly overexpress ChoK and that this expression is homogeneous throughout the tumor. We will employ intraoperative NIR fluorescence-guided surgery using our ChoK probes for detection of tumors and identification of tumor margins. After resection and recovery, canine patients will be monitored over time for tumor recurrence and patient survival. These data will be compared to positive and negative control cohorts where tumors were resected using the non-targeted fluorophore indocyanine green (ICG) or resected without the aid of NIR fluorescence guidance. These studies will test the ability of our ChoK sensors to detect tumor margins in spontaneously occurring lung cancers in a canine patient cohort. Our long-term goal is to develop targeted NIR fluorophores for real time intraoperative surgery in human cancer patients.